A semiconductor for measuring biological interactions

ABSTRACT

An apparatus and method are disclosed for electrically directly detecting biomolecular binding in a semiconductor. The semiconductor can be based on electrical percolation of nanomaterial formed in the gate region. In one embodiment of an apparatus, a semiconductor includes first and second electrodes with a gate region there between. The gate region includes a multilayered matrix of electrically conductive material with capture molecules for binding target molecules, such as antibody, receptors, DNA, RNA, peptides and aptamer. The molecular interactions between the capture molecules and the target molecules disrupts the matrix&#39;s continuity resulting in a change in electrical resistance, capacitance or impedance. The increase in resistance, capacitance or impedance can be directly measured electronically, without the need for optical sensors or labels. The multi-layered matrix can be formed from a plurality of single-walled nanotubes, graphene, or buckeyballs or any kind of conductive nanowire, such as metal nanowires or nanowires made from conductive polymers.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/115,846, filed Nov. 18, 2008, which is incorporated herein byreference.

FIELD

The present application relates to semiconductors, and, particularly, toelectrical detection of biomolecules in a semiconductor.

BACKGROUND

Biological semiconductors (BSC) are electronic components that changeconductivity based upon biological interactions, such as protein-proteininteractions, DNA-protein binding, nucleic acid binding, andhormone-receptor binding. The ability to directly measure suchbiological interactions has scientific, medical, and industrialapplicability.

Nanomaterials are increasingly being adapted for biosensing. Once suchnanomaterial can be fabricated using single-walled carbon nanotubes(SWNT). The SWNTs are molecular wires with unique electrical propertiesattractive for solid-state nanoelectronics including logic gates,digital memory, digital switching and integration into logic circuitstransistor arrays. Individual SWNTs are quantum wires so theirconductivity depends on how conduction electrons interact with the atomswithin the SWNTs. The electrical conductance of a single nanotube wasshown to be highly sensitive to its environment, and variessignificantly with changes in electrostatic charges and surfaceadsorption of many molecules. Using chemical vapor deposition (CVD) togrow individual tubes, it was shown that there is a large conductancechange in response to the electrostatic, chemical and biologicalmolecules when they are utilized as gates for field-effect transistors(FETs) chemical and biological sensors. In addition to thissemiconductor effect for an individual tube, SWNTs interconnected in asubmonolayer network (also fabricated by CVD) were shown to exhibitsemiconductor-like behavior in which the conductance can be gated andsurface interactions with biomolecules can be used for biosensing.

Unfortunately, the method of FET fabrication using individual orsubmonolayer networks of SWNTs is complex and requires on-chip SWNTssynthesis for each FET, making it particularly difficult for fabricatingmulti-FET chips. Additionally, CVD fabrication is expensive and requiresspecial expertise. For these reasons, fabricating FETs using SWNTs hasbeen very limited.

It is desirable, therefore, to provide a biological semiconductor thatis relatively simple to fabricate (especially for multi-gate devices)and can be made at low cost.

SUMMARY

An apparatus and method are disclosed for electrically detectingbiomolecular binding in a semiconductor. The apparatus and method takeadvantage of a physical principle called “electrical percolation,” whichrelates to the flow of electricity through a random resistive network.The passage of current through the network depends on the network'scontinuity, which can be varied based on the detection and/or quantityof an analyte in a biological sample.

In one embodiment of an apparatus, a semiconductor includes first andsecond electrodes with a gate region there between. The gate regionincludes a multi-layered network of electrically conductive materialwith capture biomolecules for binding target biomolecules. The networkcan be within a three-dimensional matrix. This allows capturebiomolecules to be positioned internally within the matrix and on anouter surface of the matrix. The molecular interactions between thecapture biomolecules and the target biomolecules within the matrixdisrupts the network's continuity resulting in increased electricalresistance, capacitance or impedance. Such changes in continuity can bedirectly measured using electrical resistance sensors (e.g., ohm meter)or any other electrical sensors for measuring voltage, current,capacitance or impedance, for direct detection without the need foroptical sensors or labels. Thus, biomolecular binding (e.g.,antibody/antigen) changes the conductive properties of a semiconductor,which allows for a simple and direct mechanism for detection ofbiomolecular interactions.

The multi-layered network can be formed by depositing of a variety ofcarbon-based materials, such as carbon nanotubes (CNT), graphene, orbuckeyballs or using any metallic nanowires or conductive polymernanowires. The semiconductors can be fabricated at or near thepercolation threshold. At the percolation threshold, small changes inthe molecular complexes can result in large changes in conductivityincreasing the sensitivity of detection.

In one embodiment of a method of use, biomolecular interactions aredetected using the semiconductor. A sample (such as a liquid sample) canbe introduced, which can include target biomolecules to be detected. Ifthe target biomolecules present in the sample are introduced into thesemiconductor, a binding pair is formed between a capture biomoleculeand the target biomolecule. The binding pair changes a resistance in thegate region of the semiconductor by disrupting the continuity of thenetwork. An automatic measurement of resistance can then be performed inorder to detect the biomolecular interactions. A quantitativedetermination of biomolecular activity can be made based on a comparisonbetween the measured resistance and a control resistance measurement.

In one embodiment of a method of manufacture, a semiconductor isfabricated by immobilizing capture molecules on surfaces of electricalconductors. A solution is created that is used to form a gate region ofthe semiconductor. Electrodes are then deposited on opposing sides ofthe gate region. The resultant semiconductor can then operate as atransistor.

The apparatus and method provide several advantages. First, many(similar or different) biological semiconductors can be easily depositedon the same surface enabling simultaneous multi-sample (e.g., analyzingmultiple patients for the same target) or multi-target (e.g., analyzingthe same patient for multiple different targets) analysis on the samechip. The biological semiconductor does not require specializedfabrication facilities or experience, which lowers the overall cost andbroadens the practical applications in which the semiconductors can beused. Furthermore, the semiconductor can be stored for long periods oftime before use because of its stability. Finally, the semiconductoroffers fast, continuous and nearly instantaneous detection of biologicalactivity.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show schematic profile views of a semiconductor in afirst state prior to injection of target biomolecules and a second statewith captured target biomolecules disrupting conductivity of thesemiconductor. The semiconductor includes a network of carbon nanotubesthat form a three-dimensional matrix having width, depth, and length.Specific binding molecules are present on the nanotubes throughout thematrix (i.e., interior and exterior) for binding the targetbiomolecules. Although only two dimensions are shown in FIGS. 1A and 1B,it is understood that the network also extends perpendicular to thepage. The disruption of the network is illustrated between FIG. 1A(prior to binding of the target) and FIG. 1B (after binding of thetarget) when the three-dimensional continuity of the network isdisrupted to increase resistance.

FIG. 2 shows an exemplary mold having a blank top portion and a bottomportion with a circuit including sixteen semiconductors fabricated inparallel.

FIG. 3 shows a system for continuous monitoring of a circuit includingor more biological semiconductor.

FIG. 4 is a flowchart of a method for detecting biomolecularinteractions in the semiconductor.

FIG. 5 illustrates additional process features that can be performed.

FIG. 6 is a flowchart of a method for fabricating the semiconductor ofFIG. 1.

FIG. 7 shows data from example injections of target biomolecules into aplurality of biological semiconductors, illustrating changes inresistance of the network.

FIGS. 8A and 8B illustrate percolation curves of a single-walled carbonnanotube and signal-to-baseline levels versus concentration levels ofsingle-walled carbon nanotubes according to a first example.

FIG. 9 shows signal-to-baseline levels versus measured proteins,according to the first example.

FIG. 10 is an illustration showing that quantitative measurements inFIG. 9 correspond to traditional quantitative measurement methods usinglabeling and optical sensing, according to the first example.

FIGS. 11A-C show a transistor made according to another example withoutelectrodes on the chip.

FIGS. 12A and 12B illustrate percolation curves of a single-walledcarbon nanotube according to another example.

FIG. 13 shows electrical characteristics of staphylococcal enterotoxin B(SEB) actuation of a semiconductor according to another example.

FIG. 14 shows that quantitative measurements in FIG. 13 correspond totraditional quantitative measurement methods using labeling and opticalsensing assay analysis of captured SEB on a semiconductor chip,according to a second example.

DETAILED DESCRIPTION Terms and Techniques

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided,along with the context of some of the terms in the present disclosure:

Analyte or Target: an atom, molecule, group of molecules or compound ofnatural or synthetic origin (such as, but not limited to, a drug,hormone, enzyme, growth factor antigen, antibody, hapten, lectin,apoprotein, polypeptide, cofactor) sought to be detected or measuredthat is capable of binding specifically to at least one binding partner(such as, but not limited to, a drug, hormone, antigen, antibody,hapten, lectin, apoprotein, cofactor).

The analytes may include, but are not limited to, antigens from orantibodies to infectious agents (such as HIV, HTLV, Helicobacter pylori,hepatitis, measles, mumps, or rubella), drugs (such as cocaine,benzoylecgonine, benzodiazepine, tetrahydrocannabinol, nicotine, ethanoltheophylline, phenytoin, acetaminophen, lithium, diazepam,nortriptyline, secobarbital, phenobarbitol, methamphetamine,theophylline, etc), hormones (such as testosterone, estradiol, estriol,17-hydroxyprogesterone, progesterone, thyroxine, thyroid stimulatinghormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone(LH), transforming growth factor alpha, epidermal growth factor (EGF),insulin-like growth factor (ILGF) I and II, growth hormone releaseinhibiting factor, IGA and sex hormone binding globulin); and otheranalytes including antibiotics (such as penicillin), glucose,cholesterol, caffeine, cotinine, corticosteroid binding globulin, PSA,or DHEA binding glycoprotein.

Analytes vary in size. Merely by way of example, small molecule analytescan be, for instance, <0.1 nm (such as cotinine or penicillin, each witha molecular weight of less than about 1,000 Daltons). However, analytesmay be larger, including for instance immunoglobulin analytes (such asIgG, which is about 8 nm in length and about 160,000 Daltons). Analytescan be polyvalent or monovalent. Examples of analytes are disclosed, forexample, in U.S. Pat. No. 4,299,916; U.S. Pat. No. 4,275,149; U.S. Pat.No. 4,806,311; U.S. Pat. No. 6,001,558; and PCT Publication No.98/39657.

A sample containing an analyte can be any biological fluid, such as, butnot limited to, serum, blood, plasma, cerebral spinal fluid, sputum,urine, nasal secretions, sweat, saliva, pharyngeal exudates,bronchoalveolar lavage fluids, or vaginal secretions. Fluid homogenatescan also be utilized as samples, such as cellular homogenates or fecalsuspensions. Samples can also be non-biological fluids such asenvironmental samples, plant extracts, soil extracts or water samples.Typically a sample is in a liquid or an aqueous form, or may be anaqueous extract of a solid sample.

Antibody: a protein consisting of one or more polypeptides substantiallyencoded by immunoglobulin genes or fragments of immunoglobulin genes.The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as themyriad immunoglobulin variable region genes. Light chains are classifiedas either kappa or lambda. Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, which in turn define the immunoglobulinclasses, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

Antibodies can exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H) —C_(H) 1 by adisulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting theF(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially anFab with part of the hinge region (see, Fundamental Immunology, W. E.Paul, ed., Raven Press, N.Y., 1993). While various antibody fragmentsare defined in terms of the digestion of an intact antibody, it will beappreciated that Fab′ fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody as used herein also includes antibody fragments either producedby the modification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Embodiments of the assay that useantibodies can use any form of the antibodies, such as the intactimmunoglobulin or fragments thereof that retain desired specific bindingcharacteristics.

Antibodies can be monoclonal or polyclonal, but often will bemonoclonal. Merely by way of example, such monoclonal antibodies can beprepared from murine hybridomas according to the classical method ofKohler and Milstein (Nature 256:495-497, 1975) or derivative methodsthereof. Briefly, a mouse is repetitively inoculated with a fewmicrograms of the selected analyte compound (or a fragment thereof) overa period of a few weeks. In some instances, it will be beneficial to usean adjuvant or a carrier molecule to increase the immunogenicity and/orstability of the analyte in the animal system. The mouse is thensacrificed, and the antibody-producing cells of the spleen isolated. Thespleen cells are fused by means of polyethylene glycol with mousemyeloma cells, and the excess un-fused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as ELISA,as originally described by Engvall (Meth. Enzymol. 70:419-439, 1980),and derivative methods thereof. Selected positive clones can be expandedand their monoclonal antibody product harvested for use. Detailedprocedures for monoclonal antibody production are described in Harlowand Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).

Monoclonal antibodies to different analytes are commercially available.For instance, a monoclonal antibody to estriol-3 is produced byFitzgerald Industries International (Concord, Mass.; Cat. #10-E37, Clone#M612039); likewise, Omega Biological, Inc. (Bozeman, Mont.) produces amonoclonal antibody to methamphetamine (Cat. #100-11-183, Clone Met 2).Rabbit anti-SEB can be purchased from Toxin Technology (Sarasota, Fla.).

Antigen: a chemical or biochemical structure, determinant, antigen orportion thereof that is capable of inducing the formation of anantibody.

Avidin/Streptavidin: The extraordinary affinity of avidin for biotinallows biotin-containing molecules in a complex mixture to be discretelybound with avidin. Avidin is a glycoprotein found in the egg white andtissues of birds, reptiles and amphibia. It contains four identicalsubunits having a combined mass of 67,000-68,000 daltons. Each subunitconsists of 128 amino acids and binds one molecule of biotin. Extensivechemical modification has little effect on the activity of avidin,making it especially useful for protein purification.

Another biotin-binding protein is streptavidin, which is isolated fromStreptomyces avidinii and has a mass of 60,000 daltons. In contrast toavidin, streptavidin has no carbohydrate and has a mildly acidic pI of5.5. Another version of avidin is NeutrAvidin Biotin Binding Protein(available from Pierce Biotechnology) with a mass of approximately60,000 daltons.

The avidin-biotin complex is the strongest known non-covalentinteraction (Ka=10¹⁵ M⁻¹) between a protein and ligand. The bondformation between biotin and avidin is very rapid, and once formed, isunaffected by extremes of pH, temperature, organic solvents and otherdenaturing agents.

Although examples disclosed herein use streptavidin (SA) as a specificbinding agent, the streptavidin could be substituted with other types ofavidin. The term “avidin” is meant to refer to avidin, streptavidin andother forms of avidin that have similar biotin binding characteristics.

Binding affinity: a term that refers to the strength of binding of onemolecule to another at a site on the molecule. If a particular moleculewill bind to or specifically associate with another particular molecule,these two molecules are said to exhibit binding affinity for each other.Binding affinity is related to the association constant and dissociationconstant for a pair of molecules, but it is not critical to theinvention that these constants be measured or determined. Rather,affinities as used herein to describe interactions between molecules ofthe described methods and devices are generally apparent affinities(unless otherwise specified) observed in empirical studies, which can beused to compare the relative strength with which one molecule (such asan antibody or other specific binding partner) will bind two othermolecules (such as an analyte and an analyte-tracer conjugate). Theconcepts of binding affinity, association constant, and dissociationconstant are well known.

Binding domain: the molecular structure associated with that portion ofa receptor that binds ligand. More particularly, the binding domain mayrefer to a polypeptide, natural or synthetic, or nucleic acid encodingsuch a polypeptide, the amino acid sequence of which represents aspecific (binding domain) region of a protein, which either alone or incombination with other domains, exhibits specific bindingcharacteristics that are the same or similar to those of a desiredligand/receptor binding pair. Neither the specific sequences nor thespecific boundaries of such domains are critical, so long as bindingactivity is exhibited. Likewise, used in this context, bindingcharacteristics necessarily includes a range of affinities, aviditiesand specificities, and combinations thereof, so long as binding activityis exhibited. The capture biomolecules disclosed herein may bind abinding domain of a target analyte.

Binding partner: any molecule or composition capable of recognizing andspecifically binding to a defined structural aspect of another moleculeor composition. Examples of such binding partners and correspondingmolecule or composition include antigen/antibody, hapten/antibody,cellular receptor/ligand, lectin/carbohydrate, apoprotein/cofactor andbiotin/avidin (such as biotin/streptavidin). The term “specificallybinds”, when referring to a binding partner (e.g., protein, nucleicacid, antibody, etc.), refers to a reaction that is determinative of thepresence and/or identity of one or other member of the binding pair in amixture of heterogeneous molecules (e.g., proteins and other biologics).Thus, for example, in the case of a receptor/ligand binding pair theligand would specifically and/or preferentially select its receptor froma complex mixture of molecules, or vice versa. An enzyme wouldspecifically bind to its substrate, a nucleic acid would specificallybind to its complement, an antibody would specifically bind to itsantigen. Other examples include, nucleic acids that specifically bind(hybridize) to their complement, antibodies specifically bind to theirantigen, and the like. The binding may be by one or more of a variety ofmechanisms including, but not limited to ionic interactions, and/orcovalent interactions, and/or hydrophobic interactions, and/or vanderWaals interactions, etc.

Biological interaction: A specific binding interaction that could ordoes occur in or with a living cell. For example, a biologicalinteraction includes any interaction between binding pairs, such asprotein binding (e.g., protein-protein binding or nucleic acid-proteinbinding), nucleic acid binding (e.g., protein-DNA, DNA-DNA, DNA-RNA,etc.), cellular receptor binding (e.g., a cell surface receptor orintracellular receptor that binds to a cellular ligand, such as ahormone).

Biotin binding protein: A protein (such as a specific binding protein)that binds biotin with sufficiently great affinity for an intendedpurpose. Examples of biotin binding proteins are well known in the art,and include avidin, streptavidin, NeutrAvidin, and monoclonal antibodiesor receptor molecules that specifically bind biotin.

Capture biomolecule: An organic molecule that is capable of productionsubstantially by a living cell, and can specifically bind a targetbiomolecule by a biological interaction. Capture biomolecules canundergo non-cellular modifications (such as addition of functionalgroups) that cannot be added in a cell and still be considered abiomolecule. Example capture biomolecules include antibodies, nucleicacid molecules, aptamers, peptides and receptors. In particularlydisclosed embodiments, the capture biomolecules may change conformationwhen binding to the target, and this change in conformation can disruptthe continuity of the network of nanotubes to result in increasedresistance. This increase in resistance indicates the presence of theanalyte, and the increase in resistance can in some embodiments beproportional to the amount (such as concentration) of target analytepresent in the sample.

Carbon nanotube: As used herein, the terms “carbon nanotube” and theshorthand “nanotube” refer to carbon fullerene, a synthetic graphite,which typically has a molecular weight between about 840 and greaterthan 10 million grams/mole. The carbon nanotubes can be single-walledcarbon nanotubes (SWCNT or SWNT) or multi-walled carbon nanotubes (MWCNTor MWNT). The present disclosure is not limited to any one method bywhich to produce carbon nanotubes. Rather, any suitable method can beused to produce carbon nanotubes for use in conjunction with methods andapparatus of this disclosure. Additionally, any size of carbon nanotubecan be used. Carbon nanotubes suitable can have average diameters in therange of about 1 nanometer to about 25,000 nanometers (25 microns).Alternatively, the carbon nanotubes suitable can have average diametersin the range of about 1 nanometer to about 10,000 nanometers, or about 1nanometer to about 5,000 nanometers, or about 3 nanometers to about3,000 nanometers, or about 7 nanometers to about 1,000 nanometers, oreven about 15 nanometers to about 200 nanometers. Alternatively, carbonnanotubes can have an average diameter of less than 25,000 nanometers,or less than 10,000 nanometers, or even less than 5,000 nanometers.Alternatively, carbon nanotubes suitable can have average diameters ofless than 3,000 nanometers, or less than about 1,000 nanometers, or evenless than about 500 nanometers.

The length of the carbon nanotubes is not critical and any length can beused. For example, carbon nanotubes can have lengths in the range ofabout 1 nanometer to about 25,000 nanometers (25 microns), or from about1 nanometer to about 10,000 nanometers, or about 1 nanometer to about5,000 nanometers, or about 3 nanometers to about 3,000 nanometers, orabout 7 nanometers to about 1,000 nanometers, or even about 10nanometers to about 500 nanometers. Alternatively, the carbon nanotubescan have a length of at least about 5 nanometers, at least about 10nanometers, at least about 25 nanometers, at least about 50 nanometers,at least about 100 nanometers, at least about 250 nanometers, at leastabout 1,000 nanometers, at least about 2,500 nanometers, at least about5,000 nanometers, at least about 7,500 nanometers, at least about 10,000nanometers, or even at least about 25,000 nanometers. Still further, thecarbon nanotubes can have lengths that would not be considered to benano-scale lengths.

Still further, any kind of conductive nanowire can be used. Examplenanowires include, but are not limited to metal nanowires, such as goldand silver or conductive polymers, such as polypyrolle, polythiophene,etc.

Detect or determine an analyte: An analyte is “detected” when itspresence is ascertained or discovered. “Determination” of an analyterefers to detecting an amount/concentration (either approximate orexact) of the analyte. Hence “detection” is a generic term that includeseither ascertaining its presence or determining an amount/concentration(since determining an amount can also indicate the presence of theanalyte). Embodiments of the device and method disclosed herein arecapable of detecting the presence or determining a quantity of theanalyte in a sample.

Electrical conductors: Electrical conductors are capable of allowingelectrical charges, such as electrons, to move relatively freely alongthe conductor. Example electrical conductors include carbon nanotubes,graphene, and buckyballs.

Electrical Percolation: Electrical percolation is used to characterizechanges in the connectivity of elements within the network. Electricalpercolation can be modeled as the flow of electricity through a randomlydistributed network of conducting elements. In such a network, sites(vertices) or bonds (edges) are established by randomly placingresistors in a 3-D vector space with a statistically independentprobability (p) of making contacts. At a critical threshold (pc),long-range connectivity within the vector space first appears (known asthe “percolation threshold”). Beyond this threshold, the conductingelements increase precipitously and there is an onset of a sharp andvery significant increase in the electrical conductivity of thematerial. Therefore, it is characteristic of the minimal concentrationof conductive filler required to form a randomly distributed networkthat spans the whole material system. The concentration of conductivefiller correlating to the percolation threshold will be affected, not bythe mobility of electrons within the filler, but rather by thecharacteristics that control the number of contacts and the contactresistance between filler elements. Thus, the principles governing thepercolation threshold are not “electrochemical”, but rather“electrophysical” (e.g., morphology, scale, and orientation of thefiller).

Field-Effect Transistor (FET): An FET is a type of transistor commonlyused for weak-signal amplification (for example, for amplifying wirelesssignals). In the FET, current flows along a semiconductor path calledthe channel. At one end of the channel, there is an electrode called thesource. At the other end of the channel, there is an electrode calledthe drain. The physical diameter of the channel is fixed, but itseffective electrical diameter can be varied by the application of avoltage to a control electrode called the gate. The conductivity of theFET depends, at any given instant in time, on the electrical diameter ofthe channel. A small change in gate voltage can cause a large variationin the current from the source to the drain, which is how amplificationof signals occurs. When nanowires are used to manufacture FETs, thenanowires must be positioned in a well-defined pattern and orientation,which requires specialized manufacturing techniques.

Immunogen: a chemical or biochemical structure, determinant, antigen orportion thereof, that elicits an immune response, including, forexample, polylysine, bovine serum albumin and keyhole limpet hemocyanin(KLH).

Matrix: A three-dimensional region that contains the three-dimensionalnetwork of electrical conductors. The matrix can have athree-dimensional shape and can have an irregular structure. Capturebiomolecules can be positioned throughout the matrix including oninterior conductors and exterior conductors. In some embodiments, thecapture biomolecules can be uniformly distributed throughout the width,length, and depth of the matrix.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Non-naturallyoccurring synthetic analogs include, for example and without limitation,phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs),and the like. Such polynucleotides can be synthesized, for example,using an automated DNA synthesizer. The term “oligonucleotide” typicallyrefers to short polynucleotides, generally no greater than about 50nucleotides. It will be understood that when a nucleotide sequence isrepresented by a DNA sequence (i.e., A, T, G, C), this also includes anRNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.Hence, an antisense sequence can be used as a capture biomolecule tospecifically bind a target nucleic acid molecule.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds, for example γ amide bonds(for example from the γ position of a glutamic acid side chain) or aamide bonds. When the amino acids are alpha-amino acids, either theL-optical isomer or the D-optical isomer can be used, for exampleD-glutamic acid to form poly-γ-D-glutamic acid (γDPGA). The terms“polypeptide” or “protein” as used herein is intended to encompass anyamino acid sequence and include modified sequences such asglycoproteins. The term “polypeptide” is specifically intended to covernaturally occurring proteins, as well as those that are recombinantly orsynthetically produced.

The term “polypeptide fragment” refers to a portion of a polypeptidewhich exhibits at least one useful epitope. The term “functionalfragments of a polypeptide” refers to all fragments of a polypeptidethat retain an activity of the polypeptide. Biologically functionalfragments, for example, can vary in size from a polypeptide fragment assmall as an epitope capable of binding an antibody molecule to a largepolypeptide capable of participating in the characteristic induction orprogramming of phenotypic changes within a cell.

Sample or Specimen: any cell, tissue, or fluid from a biological source(a “biological sample”), or any other medium, biological ornon-biological, that can be evaluated in accordance with the invention,such as serum or water. A sample includes, but is not limited to, abiological sample drawn from an organism (e.g. a human, a non-humanmammal, an invertebrate, a plant, a fungus, an algae, a bacteria, avirus, etc.), a sample drawn from food designed for human consumption, asample of blood destined for a blood supply, a sample from a watersupply, or the like. One example of a sample is a sample drawn from ahuman or animal to determine the presence or absence of a specificnucleic acid sequence.

A “sample suspected of containing” a particular component means a samplewith respect to which the content of the component is unknown. Forexample, a fluid sample from a human suspected of having a disease, suchas an infectious disease or a non-infectious disease, but not known tohave the disease, defines a sample suspected of containing an infectiouspathogen. Alternatively, the sample is one being analyzed for scientificresearch. “Sample” in this context includes naturally-occurring samples,such as physiological samples from humans or other animals, samples fromfood, livestock feed, etc. However, the sample can also be a productmade in a research laboratory. Typical samples taken from humans orother animals include tissue biopsies, cells, whole blood, serum orother blood fractions, urine, ocular fluid, saliva, cerebro-spinalfluid, fluid or other samples from tonsils, lymph nodes, needlebiopsies, etc. However, in particular examples disclosed herein, thesamples are liquid samples.

Specific binding partner: a member of a pair of molecules that interactby means of specific, non-covalent interactions that depend on thethree-dimensional structures of the molecules involved. Exemplary pairsof specific binding partners include antigen/antibody, hapten/antibody,ligand/receptor, nucleic acid strand/complementary nucleic acid strand,substrate/enzyme, inhibitor/enzyme, carbohydrate/lectin, biotin/avidin(such as biotin/streptavidin). Examples include a hormone binding toreceptors, and virus/cellular receptor. The methods and devicesdisclosed herein can be used for any analyte for which a specificbinding partner exists.

The phrase “specifically binds to an analyte” (or “specificallyimmunoreactive with” when referring to the particulars example of anantibody) refers to a binding reaction which is determinative of thepresence of the analyte in the presence of a heterogeneous population ofmolecules such as proteins and other biologic molecules. A cellularreceptor is, for example, capable of specifically binding to an analyte.In immunoassay conditions, the specified antibodies bind to a particularanalyte and do not bind in a significant amount to other analytespresent in the sample. A variety of immunoassay formats may be used toselect antibodies specifically immunoreactive with a particular analyte.For example, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane, Antibodies, A Laboratory Manual, CSHP, New York (1988),for a description of immunoassay formats and conditions that can be usedto determine specific immunoreactivity.

Target analyte: An analyte to be detected by the detector, and whichbinds to the specific binding partner on the nanotube.

Three-dimensional network: A complex, interconnected group of electricalconductors allowing electrical charge to pass between two points usingmultiple and unique electrical paths. For example, the network is anunpatterned, random interconnection of electrical conductors. If anyelectrical paths in the network are disrupted, electrical charge canstill pass between the two points using alternative electrical paths inthe network. The three-dimensional network can be any size (i.e., anylength, depth, and width), depending on the application. One example canuse carbon nanotubes of at least 0.4 nm in diameter. The desired depthand width of the network can be greater than a single nanotube, such as2, 3, 4, 5, etc. times the thickness of a single nanotube. Otherthicknesses can be between 10 to 100 times the thickness of a singlenanotube.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. The term“comprises” means “includes.” It is further to be understood that allmolecular weight or molecular mass values are approximate, and areprovided for description. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of this disclosure, suitable methods and materials are describedbelow. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The disclosure is illustrated by the following non-limiting Examples.

FIG. 1A shows a biological semiconductor 10 having opposing electrodes12, 14 with a gate region 16 positioned there between. The gate region16 includes a matrix 18 of electrical conductors 20 that create aplurality of electrical connections between the electrodes 12, 14. Theelectrical conductors form a three-dimensional network of individualentangled or overlapping conductors. A plurality of capture biomolecules22 are shown as half-moon shapes and are positioned within the matrix18. The capture biomolecules 22 are shown connected to the exteriorsurface of the carbon nanotubes, but are contained substantially withinthe interior region of the matrix of entangled conductors. The matrix 18is a multi-layer, three-dimensional network that can be made of avariety of conductive materials, such as carbon nanotubes (e.g.,single-walled carbon nanotubes), graphene, buckyballs, or otherfullerene material. Other materials can also be used, such as metalnanowires, made of gold, silver, etc. or conductive polymers, such aspolypyrolle, polythiophene, etc. By multi-layered, it is meant thatmultiple of the electrical conductors 20 are interconnected in thenetwork in three-dimensional space. The matrix 18 can be a solid or asolution, depending on the application. The electrodes 12 are made of aconductive metal, such as silver, but other conductive metals can beused. The capture biomolecules 22 are capable of binding/interactingwith target molecules, such as antibodies, nucleic acid molecules,aptamers, peptides, etc. The electrical conductors 20 are bound in a wayas to not overly confine the conductors to allow movement in response tobiomolecular binding between the capture biomolecules and targets, whichare introduced into the gate region 16. Although not shown, the gateregion 16 can be protected or shielded from the outside environmentusing a polymer-based coating or layer, such as ofpolydiallyldimethylammonium chloride or other suitable coveringmaterial. Such a covering material limits any surface interactionoccurring in the gate region 16, which could impact results. Thespecimen can interact with the gate region by adding the specimen priorto placing a covering material or by placing the gate in a flow cell andinjecting the specimen in the flow cell.

The opposing electrodes 12, 14 are electrically coupled to a resistancemeasuring device 24 for reading a resistance of the biologicalsemiconductor across the gate region 16. For example, the resistancemeasuring device can be a commercially available ohm meter.

The concentration of conductive material in the matrix 18 is such thatthe material is at or near a percolation threshold. The conductivity ofthe semiconductor 10 depends on the number of contacts in the networkbetween the electrical conductors 20. This number of contacts can bevaried through molecular interactions, which changes the spacingorientation, and continuity of the matrix 18. The binding of targetanalyte to the receptors within the matrix disrupts the pre-bindingarchitecture of the matrix to change the conductivity of the matrix. Asa result, the molecular interactions also change the resistance of thematrix 18, which can be used to indicate the presence and/or number ofmolecular interactions. A quantitative change in resistance can be madeto correlate with a particular quantity (such as a concentration of thetarget molecule).

FIG. 1B shows target molecules 26 bound to the capture biomolecules 22.As can be seen, the molecular interactions between the capturebiomolecules and the target molecules disrupt the matrix continuityresulting in increased resistance. The molecular interaction can includebinding of antigens to antibodies, nucleic acid binding, hormone bindingto a receptor, etc. Because the conductors 20 are not overly confined toallow for movement, some electrical paths between the electrodes 12, 14break, which forces current to pass through other conductors, increasingoverall resistance. Thus, FIG. 1A represents a low-resistance mode ofoperation. In a particular example, single-walled nanotubes were used(shown in black lines) with no antigens bound to antibodies (shown ashalf-moon shapes). By contrast, FIG. 1B represents a high-resistancemode wherein binding of antigens (ovals) results in disruption of thethree-dimensional matrix (non-contact SWNTs are shown in grey) thusincreasing electrical resistance. Disruption of the three-dimensionalmatrix means that some of the conductive paths between electrodes havebeen severed, while other conductive paths remain intact.

FIG. 1A and 1B, therefore, show that the semiconductor is based onelectrical percolation, rather than FETs where channel width changes asa result of gate activity. Semiconductors based on electricalpercolation are easier to manufacture than FETs, as there is no need fordirect chemical vapor deposition or specialized expertise needed for FETmanufacture. FETs require specific patterned, structured placement ofnanowires. By contrast, semiconductors formed using the techniquesdescribed herein use an unoriented, unstructured, pattern less,mesh-like network of interconnected conductors. The binding moleculesare therefore distributed throughout the interior of the gate region,instead of being confined to an outer surface. Unlike FET's, variousbinding partners can be used to functionalize pre-made SWNT gates inbulk. The gates can then be simply printed or deposited ontonon-conductive materials.

FIG. 2 shows a tray 40 that can be used to assemble a plurality ofbiological semiconductors in parallel. The tray acts as a base substrateand can be made of plastic or other non-conductive material. An upperportion 42 of the tray is blank and has a plurality of blank forms 44for making biological semiconductors. The forms can be any shape anddimensions. A lower portion 46 has sixteen biological semiconductorscoupled in parallel, such as a biological semiconductor shown at 47.Although sixteen are shown, the number of biological semiconductors canbe any desired number simply by modifying the size of the tray 40.Alternatively, the biological semiconductor can be manufactured as astand-alone device, such as a more traditional transistor package orchip. Positive electrodes 12 are floating and are used to connect apositive lead of an ohm meter or other resistance measuring device.Negative electrodes 14 are coupled together through an electrical common56 that extends the width of the tray. Any number of the electrodes 50can be coupled to one or more resistance measuring devices. Between thepositive and negative electrodes 12, 14 are the gate regions 16 having athree-dimensional network into which one or more different specimens maybe injected. Upon injection, molecules of the specimen can bind withcapture biomolecules in the network resulting in increased resistanceacross a gate region of the biological semiconductor. Some of thebiological semiconductors in the tray 40 can act as control elements,which are used as a baseline to show the resistance of the biologicalsemiconductors without any molecular reactions. Such a baseline isneeded in order to determine a difference between a measurement ofresistivity of a biological semiconductor that had a detectable targetmolecule and a biological semiconductor that had no specimen introducedor that had limited or no molecular binding. One example of how to usethe biological semiconductors is that each biological semiconductor canhave a different antibody associated with it. A user's sample can beinjected into all sixteen gate regions and sixteen different readingscan be taken to determine different antigens in the sample.

In the particular example of FIG. 2, the gate regions 16 are circular incross section (i.e., cylindrical in three-dimensions), but other shapescan be used, such as rectangular, square, etc.

FIG. 3 shows a system 70 for implementing continuous monitoring. One ormore biological semiconductors 72 include networks with capturebiomolecules to which targets are bound. An electrical resistancedetector 76 continuously monitors one or more of the biologicalsemiconductors in order to detect molecular interactions. The resistancedetector can be an ohm meter or other devices for measuring current,voltage, resistance capacitance or impedance. The detector 76 is coupledto a computer 78 that monitors and stores readings from the detector.Periodic readings can be taken and compared to control readings where nobiomolecular activity occurred. The computer 78 can be coupled to adigital-to-analog converter 80, which allows the computer 78 tocommunicate with a pump 82 and a plurality of valves 84. The valves 84can be coupled to one or more specimens 86, which are samples to beinjected into the biosemiconductors in order to detect biomoleculestherein. If two-way communication is desired between the computer 78 andthe pump and valves, an analog-to-digital converter can be housedtogether with the digital-to-analog converter, as is shown at 80. Suchtwo-way communication may be desirable to monitor that state of thevalves and the pumping mechanism. In general operation, the computerreleases one or more of the specimens 86 by controlling the valves 84.The pump 82 then pumps the specimen released by the valves and injectsit into the gate region 16 of the semiconductor for analysis. Using thesystem 70, biomolecular sampling and detection can be fully automated.

FIG. 4 is a flowchart of a method for detecting and measuringbiomolecules in a specimen. In process block 100, a circuit is providedincluding a biosemiconductor with a gate region of electricallyconductive material whose resistance changes with biomolecular binding.An example semiconductor is shown in FIG. 1 and example circuits areshown in FIGS. 1, 2 and 3. In process block 102, a specimen is appliedto the circuit including target biomolecules to be detected by thecircuit. For example, automatic application can occur using the systemof FIG. 3. Alternatively, manual application can be used. In processblock 104, automatic measurement of resistivity can be performed, suchas using resistance detector 76 of FIG. 3. Using the measuredresistance, a change is resistivity as compared to a previousmeasurement can be performed.

FIG. 5 shows a flowchart of a method showing additional processes thatcan be performed in conjunction with the processes of FIG. 4. In processblock 110, the specimen can be automatically injected into the gateregion 16 of the semiconductor. Such automatic injection can occurthrough using a pump 82 (FIG. 3), which is responsive to a computer 78,for pumping the specimen released by control valves 84 and deliveringthe specimen to the gate region 16 of a semiconductor. In process block112, an automatic comparison is made between the measured resistance anda control resistance. Such automatic comparison can occur using thecomputer 78. For example, the computer 78 can measure a controlresistance obtained through measuring the resistance of a biologicalsemiconductor that does not have molecular binding pairs as describedabove. In process block 114, using a difference calculation between themeasured resistance and a control resistance, a quantity of biomoleculesin the specimen can be determined.

FIG. 6 is a method of manufacturing a semiconductor for measuringbiological interactions. In process block 120, a multi-layered materialis provided that is capable of forming a network of electricalconductors. The multi-layered material can be formed from abio-nanocarbon material, such as carbon nanotubes (e.g., single-walledcarbon nanotubes), graphene, buckyballs, etc. In process block 122,capture biomolecules are immobilized onto surfaces of electricalconductors in the bio-nanocarbon material through electrostaticabsorption, using techniques well known in the art. An example of how toattach biomolecules onto bio-nanocarbon material is described in thefollowing article: Yang, M; Kostov, Y; Bruck, H; and Rasooly, A, “CarbonNanotubes with Enhanced Chemiluminescence Immunoassay for CCD-BasedDetection of Staphylococcal Enterotioxin B in Food,” AnalyticalChemistry, Vol. 80, No. 22, Nov. 15, 2008, which is hereby incorporatedby reference. Typically, the resultant multi-layered material is insolution form. In process block 124, a gate region of a semiconductor isformed by depositing the solution into a well region of a transistor. Anexample well region is shown in FIG. 2 at the center of blank form 44.The result is the gate region 16 is formed that is a three-dimensionalnetwork of electrical conductors. In process block 126, after the gateregion has been formed, electrode material (e.g., silver, gold, etc.) isdeposited at opposite ends of the gate region to create a semiconductor,such as a transistor. Alternatively, no specific electrodes are neededand the leads of the measuring device can be connected directly toopposing ends of the gates (See FIG. 11). A specific example ofmanufacture for the gate region is provided below. Additional techniquesare well known in the art. Suitable densities of nanotube material canbe derived from percolation curves associated with the material used.For example, in FIG. 8A, the density determined can be based on wherethe percolation curve levels off, such as between 0.5 and 1.5. Someexamples densities have been shown in the art and are described in U.S.Pat. No. 6,918,284, at column 2, lines 54-64, which is herebyincorporated by reference.

FIG. 7 includes graphs A, B, and C showing an experiment with the systemin continuous monitoring mode. Staphylococcal enterotoxin B (SEB) wasinjected into the biological semiconductors. Different controls wereused for nonspecific binding. The trace labeled “a” shows 100 ng/mL ofSEB. As can be seen, all traces react nearly simultaneously to injectionof target biomolecules. The trace labeled “b” shows 1 g/mL BSA. Thetrace labeled “c” shows 1 g/mL of lysozyme. And the trace labeled “d”shows 1 g/mL IgG applied to a sensor. Graph B demonstrates the rapidresponse of the biological semiconductor. Graph C demonstrates SEB isbound to the biological semiconductor by having a first injection ofSEB, shown at I, and a second injection shown at II. The signal isdetected as shown by trace “e”, but it is not detected in control trace“f.”

EXAMPLE 1 Using Single-Walled Carbon Nanotubes

A detector was fabricated with the bio-nanocomposite material bydepositing pre-functionalized single-walled nanotubess (SWNTs) withbiological ligands to form a biological semiconductor (BSC) layer.

A simplified prototype of the BSC sensor is shown in FIG. 2. The BSC isa unipolar device, with two electrodes painted with silver contact pasteon both sides of the printed SWNT-antibody bio-nanocomposite. SeveralBSCs can be easily fabricated in a row on the same surface. At thecircuit level, each BSC contains a connection well, shown at 130 for thesilver electrode, a channel, shown at 132 for the bio-nanocomposite anda channel 134 for the silver electrode that functions as a common groundpoint for all BSCs. The resultant plurality of semiconductors are shownin the lower portion 46 of FIG. 2.

The SWNTs were functionalized with rabbit anti-SEB IgG. A previouslydeveloped CNT functionalization scheme is employed for binding the SWNTswith antibodies. The fabrication and preparation of the carbon nanotubesis described in more detail in Example 2. The bio-nanocomposite is thenimmobilized by drying it directly on the surface of either Poly(methylmethacrylate) (PMMA) or polycarbonate wells fabricated by lasermicromachining.

The BSC is operated simply by measuring the electrical resistancebetween the silver paste electrodes. Binding of the specific antigen tothe antibody disrupts the network and increases the resistance. Theamount of binding of the specific antigen to the antibody controls theoverall resistance of the electrical percolation BSC network, which ismeasured by an ohmmeter via each BSC electrode 50 and the commonelectrode 56.

A circuit board with sixteen electrical percolation BSCs was used in aconventional immunodetection assay by allowing binding of SEB to theantibody gate and washing off unbound material. The measurement valuewas calculated as the difference between the initial reading recorded(R0) with no SEB and the reading with SEB (R1). The difference betweenthe two readings (R1−R0) was measured as the signal, and is normalizedby R0 to obtain the signal-to-baseline ratio. FIG. 8 includes two graphsshowing concentration levels for single-walled carbon nanotubes. Varyingconcentration levels can be used based on the application, but forsingle-walled carbon nanotubes it is believed that a concentration ofabout between 1 and 1.5 mg/ml is suitable. Whatever material is used,the concentration levels can be chosen to that the network is maintainedat around the percolation threshold so that any molecular interactionscan have a measurable impact on resistance. Using percolationprinciples, it is possible to characterize changes in the connectivityof elements within the network by modeling electrical percolation as theflow of electricity through a randomly distributed network of conductingelements. In such a network, sites (vertices) or bonds (edges) areestablished by randomly placing resistors in a 3-D vector space with astatistically independent probability (p) of making contacts. At acritical threshold (pc), long-range connectivity within the vector spacefirst appears (known as the “percolation threshold”). Beyond thisthreshold, the conducting elements increase precipitously and there isan onset of a sharp and very significant increase in the electricalconductivity of the material. Therefore, it is characteristic of theminimal concentration of conductive filler required to form a randomlydistributed network that spans the whole material system. Theconcentration of conductive filler correlating to the percolationthreshold will be affected, not by the mobility of electrons within thefiller, but rather by the characteristics that control the number ofcontacts and the contact resistance between filler elements. Thus, theprinciples governing the percolation threshold are not“electrochemical”, but rather “electrophysical” (e.g., morphology,scale, and orientation of the filler).

FIG. 8 shows an establishment of a percolation threshold of theSWNT-antibody network using various concentrations of SWNT immobilizedonto a PMMA surface without (FIG. 8A, labeled (a)) and with anti-SEBantibody (FIG. 8A, labeled (b)). Their resistance was measured todetermine the percolation threshold, vp, using a conventional power lawequation from percolation theory with a baseline resistance. Using apower law fit, it was possible to determine that the percolationthreshold for the SWNT-antibody network is between 0.2 to 0.3 mg/mL, anddoes not change significantly after antibody immobilization. The rate ofchange in resistance is directly related to the power-law exponent, n,which was 8 and the power-law coefficient, a, which was 5.×10−6, in thisparticular example. There are three characteristic regimes in SWNTconcentration associated with these values: (1) between ˜0.2 to 0.5mg/mL the percolation threshold is characterized by a steep change(approximately four orders of magnitude) in resistance due to the onsetof percolation, (2) between ˜0.5 to 1 mg/mL the change levels off andthe increase is approximately one order of magnitude, (3) over ˜1 mg/mlthe resistance levels off and does not change significantly with higherconcentrations of SWNT resulting in complete percolation. Over theentire range, the total change in resistance is approximately fiveorders of magnitude. The percolation threshold of the SWNT-antibodybio-nanocomposite network also indicates that its typical resistance(FIG. 8A, labeled (b)) will be higher than the resistance that isattributed to the SWNT only (FIG. 8A, labeled (a)), presumably due tothe contacts between the antibody and the functionalized SWNT.

At the percolation transition point, the point above the percolationthreshold where the change in resistance begins to level off, there is astill relatively low statistical distribution of “contacts” between theCNT-antibody complexes in the network. Therefore, small changes in theCNT-antibody complexes can lead to dramatic changes in conductivity.Based on this model, the bio-nanocomposite prepared with 1 mg/mL of SWNTwill be the most sensitive to molecular interactions forimmunodetection, since this is the concentration at which the change inresistance begins to level off, consistent with the complete percolationof the SWNTs.

To validate the prediction that the point where complete percolationoccurs (1 mg/mL) will be the most sensitive to molecular interactions,the response of the BSC over a range of SWNTs concentrations (0.5-3mg/ml) was analyzed in response to binding of broad range of SEBconcentration (0.5-100 ng/ml). At the transition point of 1 mg/ml, theBSC exhibited peak sensitivity to all SEB concentrations (FIG. 8B). Thisresult suggests that the mechanism of the BSC sensor is electricalpercolation. Moreover, for all concentrations of SWNTsbio-nanocomposite, the S/B increases with increasing SEB concentration,suggesting that the new BSC can be used for direct biosensing andbioactuation.

To show the specificity of the BSC response, various amounts of SEB(from 0.1-100 ng/mL) in buffer were added to the chip with 1 mg/ml ofSWNT (FIG. 9, labeled (a)). The resistance increased proportionally tothe amount of SEB. Non-specific antigens were used to study the BSC leakrate, which is the change in resistance with non-specific binding and isan indication of the specificity and the selectivity of BSC actuation.Various non-specific antigens were used, including a smaller molecularweight (14 kDa) protein, lysozyme (FIG. 9, labeled (b)), and a highermolecular weight (150 kDa) protein, human IgG (FIG. 9, labeled (c)). Asshown in FIG. 9, the level of non-specific binding in thesesemiconductors is relatively small regardless of concentration, which issimilar to the S/B for SEB concentrations when there is no antibody onthe SWNTs (FIG. 9, labeled (d)).

To determine the limit of detection (LOD) for SEB, the S/B ratio fromeight replicas of various concentrations of SEB was compared to buffer.A T-test demonstrated that at 1 ng/ml, the S/B ratio is significantlydifferent (P<0.00017) from the value using buffer only. Thus, thecurrent configuration has a LOD of 1 ng/mL for SEB.

To confirm that the percolation of the SWNT-antibody and the antibodygate mechanisms depend on SEB binding, an independent measurement ofbound SEB to the SWNTs bio-nanocomposite was carried out using asandwich immunoassay detected by Enhanced Chemiluminescence (ECL). Asshown in FIG. 10A, the intensity of the signal from the captured SEB onthe BSC chip is proportional to the amount of SEB. Quantitative analysisof the data (FIG. 10C) suggests a high correlation between the amount ofSEB and the ECL signal and that there is a very high correlation(R2=0.9942) between the electrical measurements (FIG. 9) and the ECLmeasurements (FIG. 10B). The linear regression is also highlysignificant (p<0.0056), suggesting that the anti-SEB antibody on the BSCchip did indeed capture SEB, and that the direct electrical measurementsare in agreement with the indirect sandwich immunoassay detected by ECL.

The data suggests that antigen binding leads to rearrangement of theSWNT-antibody network, resulting in physical depletion of electroncarriers in the bulk of the SWNT-antibody bio-nanocomposite throughchanges in contact between the SWNTs. Such contacts are analogous to thephysical edge of the conduction band. At this point, the antibody gatemechanism initiated by binding the antigen to the antibody shifts thecomplex closer to the band gap, which is an energy range wherestatistically few electron states exist so fewer electrons can jumpbetween SWNTs. This is analogous to decreasing an electric field in aclassical semiconductor, and therefore increases the electricalresistance of the SWNT-antibody network. The percolated SWNT-antibodynetwork can therefore be considered the “conduction band”, and thenumber of electrons in the conduction band (i.e., the band gap) isphysically determined by the number of SWNT-antibody complexes in theconduction band, rather than by the conventional electronic band gap atthe surface of the SWNT that is responsible for electrochemicaldetection principles.

Unlike field-effect transistors (FETs) based sensors, which rely on anelectric field at the surface of the SWNTs to control conductivity, theresponse of the electrical percolation BSC can be attributed to thenumber of contacts of carbon nanotubes within the network. Since thenumber of contacts can be varied by molecular interactions (i.e., byantibody-antigen binding), changes in the resistance of the network canbe used to determine the number of interactions and hence theconcentration of the target molecule.

One attractive feature of electrical percolation BSCs based on SWNTs isthe simplicity of the preparation (screen printing). In contrast, FETsare often fabricated using chemical vapor deposition (CVD) and require ahigh-tech infrastructure for microfabrication of solid-statesemiconductor components. Furthermore, unlike FETs which are constructedwith SWNTs as a single wire or sub-monolayer network, BSC do not need tobe oriented. In fact, a multi-layer mesh-like network is preferred.Electrical percolation BSCs can simply be printed on any non-conductivematerial to create biosensors capable of detecting a variety ofmolecules. Selectivity is achieved by printing different specificbiological “gates”, such as antibodies, DNA, receptors, or aptamers,taking advantage of the natural selectivity of these biologicalmolecules. Moreover, electrical percolation BSC production can bereadily scaled to perform multi-analyte detection, unlike single CNTdevices that are challenging to fabricate and functionalize.

Having simple biosensor technology may permit wider use of biosensors.Existing technologies are relatively complex, have relatively limitedcapability for multi-analyte detection, and are costly. The BSC proposedhere overcomes each of these limitations. BSCs are very simple tofabricate and to operate and are capable of multi-analyte detection.Using BSCs, it is possible to fabricate miniaturized “Biological CentralProcessing Units (CPUs)” with multiple biological elements, capable ofprocessing and sorting out information on multiple analytessimultaneously. By combining them with computer algorithms, it ispossible to automatically perform multi-analyte detection and makedecisions analogous to the way a silicon chip processes digitalinformation to make decisions important for direct biodetection ofmultiple microbial pathogens and their toxins, numerous cancerbiomarkers, cardiovascular or kidney biomarkers.

EXAMPLE 2 Fabrication of the Gate Region

Materials and Reagents: Staphylococcal enterotoxin B (SEB), rabbitanti-SEB affinity purified IgG, and peroxidase (HRP) conjugated anti-SEBIgG were purchased from Toxin Technology (Sarasota, Fla.). Single-walledCarbon Nanotubes (CNTs) were obtained from Carbon Solutions Inc(Riverside, Calif.). Poly(diallyldimethylammonium chloride) polymer(PDDA) was purchased from Sigma-Aldrich (St. Louis, Mo.). Silver contact“Silver Liquid” was purchased from Electron Microscopy Sciences(Hatfield, Pa.). For ECL detection, Immun-Star HRP Chemiluminescence Kitwas obtained from Bio-Rad (Hercules, Calif.). All other reagents were ofanalytical grade and de-ionized water was used throughout.

Fabrication of BSC sensor: The BSC sensor was designed in CorelDraw11(Corel Corp. Ontario, Canada) and micro-machined in 1.5 mm acrylic usinga computer controlled laser cutter Epilog Legend CO2 65W cutter (Epilog,Golden, Colo.). Before engraving the common electrode for all sixteenBSCs, the connection well for the readout electrode and cutting theslots for the bio-nanocomposite material, the lower side of the PMMAsheet was coated with 3M 9770 adhesive transfer doublesided tape(Piedmont Plastics, Beltsville, Md.) and the polycarbonate film wasimmobilized directly on the PMMA. The bio-nanocomposite was bonded tothe polycarbonate film and the electrodes were filled with silverconducting paste.

Carbon nanotube preparation: The CNTs (30 mg) were first shortened andoxidized by mixing with concentrated sulfuric acid and nitric acidmixture (3:1 v/v) and sonicating with a Fisher (FS-14) sonicator for 6 hfollowed by extensive washing in water (100 ml) until neutralized (pH7.0). Then the CNT were dispersed in 100 ml 1M NaOH solution for 5 minto achieve net negative charged carboxylic acid groups and washed withwater (100 ml).

CNT functionalization: a linker molecule to the carbon nanotube was usedto attach the capture biomolecule. Poly(diallyldimethylammonium chloride(PDDA) is positively charged and SEB antibody was negatively charged, soantibodies electrostatically adsorbed onto carbon nanotube. Thepositively charged polycation was adsorbed by dispersing the CNT in 50ml of 1 mg/mL PDDA containing 0.5 M NaCl for 30 min followed bycentrifugation (10,000 RPM in Beckman centrifuge for 15 minutes) andwashed with 100 ml of water.

CNT-antibody complex preparation: The CNT were functionalized bydispersing in a rabbit anti-SEB IgG phosphate buffer solution (20 mM, pH8.0) at a concentration of 0.01 mg/mL for 1 h at room temperature, sothat the antibody was adsorbed onto the CNT surface. Aftercentrifugation (15 minutes) and washing extensively with water (10 ml),the modified CNT was stored at 4° C. in pH 8.0 phosphate buffer at aconcentration of approximately 1 mg/mL for no more than two weeks beforeuse.

BSC detection of SEB: The CNT-antibody complex described above isimmobilized directly on polycarbonate. Before applying SEB samples, theresistance of the BSC is first measured (R₀). Different concentrationsof SEB samples in phosphate buffer are added to sample wells andincubated for 60 min at room temperature (25° C.). After washing, theBSC was dried at room temperature for 2 hours and the resistancemeasured again (R₁). The difference between the two readings (R₁−R₀) isused as signal corresponding to different concentration of SEB.

SEB detection using ECL: For the control experiment, after SEB binding,the BSC was then blocked with 1% BSA in 15 μl buffer for 30 min. A HRPconjugated anti-rabbit IgG was added to the captured SEB and after 60minutes incubation and washing, ECL was achieved by adding 7 mL of ECLbuffer (formed by mixing the two solutions from the chemiluminescent kitin a 1:1 volume ratio) into each well and the ECL intensity was measuredimmediately with a custom-built point-of-care CCD detector. TheCCD-based detector consists of an SXVF-M7.cooled CCD camera (AdirondackVideo Astronomy, Hudson Falls, N.Y.) equipped with a 5 mm extension tubeand a 12 mmPentax f1.2 lens (Spytown, Utopia, N.Y.). The luminescencewas measured after 2 and 10 minutes of exposure. The CCD imageintensities were analyzed using ImageJ software, developed anddistributed freely by the NIH, and the data generated was then importedinto Microsoft Excel for further manipulation.

EXAMPLE 3 Food Sample Analysis

Food sample spiking: To spike samples of soy milk, 1 mL of sample wastransferred directly to a 2 mL centrifuge tube and differentconcentrations of SEB were added.

Cation exchanger carboxymethylcellulose (CM) for partial samplepurification: To detect SEB in food samples, partial sample purificationwas used to help reduce assay background by reducing cross-reaction ofthe antibodies with other components of the food matrix. In order toestablish a widely applicable assay for different foods, the new SWNT-ELimmunoassay method was tested in food samples with and without partialtoxin purification using the cation exchanger carboxymethylcellulose(Clvl). To prepare eM for approximately 100 purification assays, 1 g ofeM was equilibrated with 20 mL loading buffer (loading buffer=5 mMNaP03, pH 5.7) until the gel was swollen and then centrifuged at 4000rrnp for 2 min. After centrifugation, the gel was allowed to settle, thebuffer was removed and the Cvl was washed twice with 20 ml of loadingbuffer followed by centrifugation as above. After the removal of thebuffer in the last centrifugation, loading buffer was added to a finalvolume of 20 mL. The equilibrated Clvl was stored at 4 ″c before use.

Sample preparation: The tubes containing the spiked food samples werevortexed briefly, centrifuged at 14,000 rpm for 2 min, and the resultingsupernatant transferred into a fresh tube. Loading buffer was added intothe supernatant to reach a sample volume of I mL. The sample (400 J.1L)was mixed with 200 J.1L of equilibrated eM, and vortexed for 30 min.which was following by centrifugation at 14,000 rpm for 2 min and thesoluble material was removed. The material left at the tube was thenwashed 3 times by repeatedly adding loading buffer and centrifuging.Finally, 100 J.1L elution buffer (50 mM aP03, pH 6.5; 50 mM NaCl) wasadded to the matrix, the tubes centrifuged and eluted material collectedfor immunoassay.

The semiconductor sensor used (See FIG. 11) is a unipolar device, withtwo electrodes on both side of the SWNT-antibody resistor printed on aPMMA surface. Several BSCs can be printed on the same PMMA surface. Atthe circuit level, semiconductor operation is simple: the current flowthrough the BSC gate via the source and drain electrodes and theapplication of the specific antigen into the gate controls theresistance of the semiconductor, which is measured by an ohm meter viathe electrodes.

The biological nanocomposite sensor acts as a semiconductor with avariable gate with an ON mode and with a constant current flow betweenthe source and drain. Upon actuation by binding of antigen to theantibody on the gate (FIG. 11B), the current flow changes with thechange of resistance of the semiconductor. Such change in conductivity(the variable OFF mode) depends on the amount of antigens bound.

A simplified prototype of the semiconductor sensor is shown in FIG. 11C.The rabbit anti-SEB IgG-functionalized SWNT gate was immobilized intothe PMMA circuit board fabricated with laser micromachining. Apreviously developed CNT functionalization scheme was used in whichshortened (by sonication) and oxidized CNT (reacted with concentratedsulfuric acid and nitric acid mixture) were dispersed in NaOH solutionto achieve net negative charged carboxylic acid groups which then absorba linker molecule Poly(diallyldimethylammonium chloride (PDDA) which ispositively charged. The activated CNT was then reacted with rabbitanti-SEB IgG which are negatively charged, so antibodies electrostatically adsorbed onto carbon nanotube.

The circuit board with eight such semiconductors was then used forconventional immunodetection assay including binding of SEB to the gateand washing unbound material. A second layer of PMMA with holes for thesource and the drain electrodes (FIG. 11C, middle portion) was used toseal the eight BSC sensor chip. The assembled chip is shown in FIG. 11C,bottom. For SEB measurement, the source and the drain electrodes wereinserted into the holes to measure change in resistance upon actuationby SEB. The measurement value was calculated as the difference betweenthe initial reading recorded (Rn) with no SEB and the reading with SEB(Rr). The difference between the two reading was used as signalcorresponding to different concentration of SEB.

SWNT-antibody percolation curve: For establishing SWNT-antibodypercolation curve, various concentrations of SWNT (FIG. 12A, labeled as(a)) and SWNT film immobilized with primary antibody (FIG. 12A, labeledas (b)) were immobilized on PMMA at various SWNT concentrations, v, andtheir resistance, Q, was measured to determine the percolationthreshold, vP′ using a conventional power law equation. The percolationthreshold for SWNT is between 0.2 to 0.3 mg/mL (FIG. 2A a), while afterantibody immobilized, the threshold shifted to between 0.3 and 0.4mg/mL, reducing the base conductivity, σ, by ⅓rd and increasing theexponent, n, from 8 to 9 while shifting the percolation threshold from0.2 to 0.22.

The percolation threshold of the SWNT-antibody indicates that thetypical resistance of the will be higher than the resistance isattributed to the SWNT, presumably due to the contacts between theantibody and the SWNT in the functionalized SWNT. The change inresistance as the SWNTs percolate is approximately 5 orders ofmagnitude. For irnmunodetection, the SWNT film was prepared with 1 mg/mLof SWNT, which has a resistance of around 1 kO, and corresponds to thepoint at which the change in resistance begins to level off, consistentwith the complete percolation of the SWNTs. For the detection of SEB,after the antibody is immobilized and the BSA blocked, the resistanceincreased to about 5 kO. Thus, this 1 mg/mL of SWNT should provide thegreatest sensitivity to the increase in resistance due to SEB.

Electrical percolation biological semiconductor immunosensor: Thehighest sensitivity to SEB for the immunosensor is around 1 mg/mL SWNT(FIG. 12(B)), which is the point at which the percolation is completeand subsequent increases correspond to the increase in SWNTconcentration. This level is determined by the amount of antibodyimmobilized on the SWNTs, which will affect the sensitivity. Uponactuation by SEB, the resistance increases, e.g. for the detection of Ing/mL SEB, resistance change is about O.5K oluns. When various amountsof SEB (50, 10, 5,1, 0.1, 0.5, 0.01, ng/mL as well as the baseline at °ng/mL) in buffer were added to the chip (FIG. 13), the resistanceincreased proportionally to the amount of SEB. To produce a calibrationcurve, a quadratic logarithmic fit was applied and was found to be thebest for the log of SEB concentration. The log fit indicates a LOD thatis −0.01 ng/mL, which is similar to typical levels of detection forcommercial colorimetric ELISA systems which is from 0.5 to 2 ng/gr offood (16, 18-20, 25, 27). For determining the reproducibility of themeasurements, eight samples of 1 ng/ml, SEB were analyzed and the RSDwas 10.5%.

To study the BSC leak rate, which is the change in resistance withnon-specific binding and is an indication of the specificity and theselectivity of BSC actuation, high concentrations of none specificantigens including smaller molecular weight (14 kDa) protein lysozyme(FIG. 13B, labeled (a)) and higher MW protein human IgO which is 150 kDaprotein were used to actuate the chip. In this experiment, highconcentrations of the non-specific antigens (e.g. up to 50 ng/ml) wereused to measure the leak rate. As shown in FIG. 13, the level ofnon-specific leak rate in these semiconductors is small and can be usedto define the BSC limit of detection.

Sandwich assay analysis of captured SEB on WPBS chip: To further analyzethe electric signal shown in FIG. 14, SEB captured on the BSC by asandwich immunoassay was measured. The chip with the captured SEBincubated with different concentrations of SEB was washed (2 mlphosphate buffer), and the immunosensor coated with antibody-antigencomplex was exposed to Horseradish Peroxidase (HRP) conjugated anti-SEBIgO (15 J1L, 0.01 mg/mL) in buffer for 1 h followed by 3 cycles ofwashing with 2 ml of phosphate buffer. HRP was assayed with ECL byadding 7 ul. of ECL buffer (mix the two solutions from ChemiluminescentKit in a 1:1 volume ratio) into each well and the ECL intensity wasmeasured immediately with a custom-built Point-of-Care CCO detectordescribed in previous work. The luminescence was measured over the fulloptical spectrum after 20 minutes of exposure. The CCO Image intensitieswere analyzed using ImageJ software, and the data generated is thenanalyzed using a concentration of 0 ng/mL SEB as background. The ratioof the signal to background (S/B) ratio was further used to quantify theSEB concentration. The intensity of the signal from the captured SEB onthe BSC chip is proportional to the amount of SEB. Quantitative analysisof the data (FIG. 14B) suggest a high correlation between the amount ofSEB and the ECL signal and that there is a very high correlation(r2=0.9996) between the electric measurements (FIG. 13) and the ECLmeasurements

The linear regression (Y=18.774S+2.0556X) is highly significant(P<O.002) suggesting that indeed the anti SEB 109 on the BSC chip indeedcaptured SEB and that the direct electrical measures are in agreementwith the indirect sandwich immunoassay.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A semiconductor, comprising: first and second electrodes; and a gateregion between the first and second electrodes, the gate regionincluding a three-dimensional matrix of electrical conductors, whereinmultiple of the electrical conductors have capture biomolecules boundthereto within the matrix that disrupt the three-dimensional matrix ofelectrical conductors when the capture biomolecules bind to a targetmolecule; the matrix of electrical conductors having a modifiableresistivity, impedance or capacitance related to molecular bindingbetween the capture biomolecules and target molecules.
 2. Thesemiconductor of claim 1, wherein the capture biomolecules include oneor more of the following: antibodies, nucleic acid molecules such as DNAor RNA, peptides such as receptors, enzymes or aptamers.
 3. Thesemiconductor of claim 1, wherein the electrical conductors in thethree-dimensional matrix are unstructured and are randomlyinterconnected.
 4. The semiconductor of claim 1, wherein thethree-dimensional matrix is formed from a plurality of single-wallednanotubes, or nanowires made of metal, such as gold or silver, orconductive polymers, such as polypyrolle and polythiophene.
 5. Thesemiconductor of claim 1, further including a substrate and wherein theelectrical conductors are bound to a surface of the substrate to allowmovement of the conductors in response to molecular binding.
 6. Thesemiconductor of claim 1, wherein electrical conductivity, capacitanceor impedance of the gate region is based on electrical percolation andthe gate region is substantially near a percolation threshold.
 7. Thesemiconductor of claim 1, wherein the matrix includes a multilayer,three-dimensional network formed from one or more of the following:carbon nanotubes, graphene, and buckyballs, metal nanowires andnanowires made from conducting polymers.
 8. The semiconductor of claim1, wherein the capture biomolecules bind with the target molecules tocreate a specific binding pair, which is selected from a group of thefollowing: antigens and antibodies, nucleic acid molecules, and hormoneand receptor.
 9. The semiconductor of claim 1, wherein the electricalconductors are nanotubes and further including shielding the nanotubesfrom the environment using a layer of polydiallyldimethylammoniumchloride.
 10. The semiconductor of claim 1, wherein the matrix ofelectrical conductors is formed by carbon nanotubes and wherein themolecular binding reduces the number of contacts between the carbonnanotubes in the matrix.
 11. The semiconductor of claim 1, wherein thefirst and second electrodes and the gate form a transistor.
 12. Thesemiconductor of claim 1, wherein the resistivity, capacitance orimpedance of the biological semiconductor can be read using the firstand second electrodes.
 13. The semiconductor of claim 1, whereinconductivity of the matrix is based on electrical percolation.
 14. Acircuit coupled to the semiconductor of claim 1 for measuring resistanceacross the semiconductor.
 15. A method of detecting molecularinteractions in a semiconductor, comprising: providing the semiconductorof claim 1; applying a specimen including target molecules to bedetected by the semiconductor; automatically measuring resistancebetween the first and second electrodes in order to detect whether thetarget molecules are bound with the capture biomolecules.
 16. The methodof claim 15, further including automatically comparing the measuredresistance to a control resistance to determine a quantity of the targetmolecules that are bound with the capture biomolecules.
 17. The methodof claim 15, wherein automatically measuring resistance includescoupling an ohm meter between the first and second electrodes.
 18. Themethod of claim 15, further including automatically injecting thespecimen into the gate region and automatically detecting whether targetmolecules of interest are in the specimen.
 19. A system for detectingmolecular interactions, comprising: a semiconductor according to claim1; a resistivity measurement device coupled to the first and secondelectrodes; a computer coupled to the resistivity measurement device forautomatically and continuously monitoring resistivity; a plurality ofvalves controlling different specimens to be supplied to the gate regionof the semiconductor, the plurality of valves coupled to the computer,which automatically controls the valves to release one or more specimensto the gate region.
 20. The system of claim 19, wherein the resistivitymeasurement device is an Ohm meter.
 21. The system of claim 19, whereinthe capture biomolecules include one or more of the following:antibodies, nucleic acid molecules, enzymes, aptamers, and peptides. 22.The system of claim 19, wherein the matrix of electrical conductors isformed by bio-nanocarbon material interconnected in a random,unpatterned fashion.
 23. The system of claim 22, wherein thebio-nanocarbon material includes a plurality of single-walled nanotubes.24. The system of claim 19, further including a pump positioned betweenthe plurality of valves and the semiconductor.
 25. The system of claim19, further including a digital-to-analog converter positioned betweenthe plurality of valves and the computer to facilitate communicationtherebetween.
 26. The system of claim 19, wherein the matrix is amultilayer, three-dimensional network formed from one or more of thefollowing: carbon nanotubes, graphene, and buckyballs.
 27. The system ofclaim 19, wherein the capture biomolecules bind with the targetmolecules to create a binding pair, which is selected from a group ofthe following: antigens and antibodies, nucleic acid molecules, andhormone and receptor.
 28. A method of manufacturing a semiconductor,comprising: providing a multi-layered material capable of forming amatrix of electrical conductors; immobilizing capture biomolecules ontosurfaces of the electrical conductors through electrostatic absorptionto form a solution; form a gate region of the semiconductor bydepositing the solution into a well region; and after the gate regionhas been formed, depositing electrode material at opposing ends of thegate region.
 29. The method of claim 28, wherein the gate region isformed on a non-conductive substrate.
 30. The method of claim 28,further including forming multiple semiconductors in parallel bycoupling electrodes at one of the opposing ends together to formed on acommon.